Figure 1.

Using internal standard-assisted synthesis and degradation mass spectrometry (iSDMS) to study the roles of EGF on global protein synthesis and degradation. SKBR3_shVPS4B and the parental SKBR3 cells were first labeled with ¹³C₆-arginine (Arg6) and D₄-lysine (Lys4) medium (labeled in red). After overnight serum starvation, Arg6/Lys4-labeled cells were stimulated with 100 ng/mL EGF in medium supplemented with regular arginine (Arg0) and lysine (Lys0) for 0, 2, 6, 12, and 24 hr (labeled in blue). Protein isolated from SKBR3_shVPS4B cells labeled with ¹³C₆ ¹⁵N₄-arginine (Arg10) and ¹³C₆ ¹⁵N₂-lysine (Lys8)—internal standard (labeled in green)—was spiked into each sample at a ratio of 1 : 3 (wt/wt). The mixtures were digested by the Filter Aided Sample Preparation (FASP) procedure, followed by strong anion exchange (SAX) peptide fractionation. Peptides were analyzed by online LC-MS/MS using an LTQ-Orbitrap mass spectrometer. The relative abundance of the newly synthesized (A _l) or preexisting peptides (A _m) was defined as the ratio of mass spectrometric peak intensities of the unlabeled peptides (I _l) or Arg6/Lys4-labeled peptides (I _m) to the intensities of the Arg10/Lys8-labeled peptides (I _h), respectively.